The Mxi1 protein (and other members of the Mad/Mxi1 family) functions as a Myc antagonist and a growth/tumor suppressor, in part by effecting transcriptional repression of key target genes. This repression has been shown to occur through Mxi1's recruitment (via its amino terminal domain) of the Sin3 proteins and their associated transcriptional repressors including histone deacetylase. Our preliminary data and a recent report suggest that Mxi1 may also bring about a different mode of repression via its carboxyl terminal domain. The objective of this proposal is to pursue this new function assigned to Mxi1's carboxyl terminal domain by defining associated factors that mediate its activities as well as gene targets that are subject to its repression. To accomplish this objective, we will first focus upon novel interactions that we have identified between the Mxi1 carboxyl terminus and (i) members of the RYBP/Yaf-2 adapter protein family and (ii) members of the Polycomb group of transcriptional repressors. Since the RYBP (and Yaf-2) adapter(s) also can recruit Polycomb group proteins, we hypothesize that Mxi1 may be a potential targeting factor for this type of transcriptional repressor that functions in multi-protein complexes to generate repressive chromatin structures. Here we will continue to characterize and expand upon these interactions and will assess their functional ramifications with respect to known properties of Mxi1. A priority will be to ascertain whether these proteins indeed mediate the transcriptional repression activity assigned to the Mxi1 carboxyl terminus to which they bind. Concomitantly, the basis for Mxi1 's harboring two distinct repression domains/mechanisms will be probed through the identification and classification of genes repressed by each or both of these domains. Taken together, our findings will forge further links between factors involved in transcriptional regulation and factors involved in the genesis/suppression of neoplastic disease. Moreover, a better understanding of the molecular functions of the Myc antagonists (e.g., Mxi1) may suggest new therapeutic approaches for the plethora of cancer types in which MYC is disregulated.